The long-term goal of the research proposed here is to understand the signaling mechanisms that organize and pattern tissues during development. We study how cells signal and respond to patterning cues, focusing on the long filopodial extensions (cytonemes) that imaginal disc and tracheal cells make. Our work has shown that these structures are present at sites of long distance cell signaling, extending from cells receiving signals and appearing to make direct contact with cells producing the signals. These observations suggest a model for selective signaling involving direct contact and regulated release and uptake. The five Specific Aims we propose are designed to help us understand the structure and function of these cell extensions and to provide new evidence relevant to our proposed model for cell-cell signaling. SPECIFIC AIM 1 Cytoneme dynamics and structure. Improved methods for light and EM microscopy will be used to image cytonemes. Better imaging will provide means to address basic questions about the growth, stability and distribution of the cell extensions. SPECIFIC AIM 2 The role of Vav in the production and function of cytoneme-like filopodia. We recently discovered that the Drosophila Rho GEF, Vav, can induce long filopodia when expressed in S2 and CI-8 cells. We will characterize the dynamics and function of these filopodia in cultured cells, and we will analyze the role of vav during imaginal disc development. We will carry out an RNAi intereference screen to identify functions that enhance or suppress filopodia formation and will examine the role of these functions during fly development. SPECIFIC AIM 3 A system to model filopodia-mediated signaling. We have developed an assay of Hh signaling in S2 cells that may be dependent upon cell-cell contact. We will work with this assay to establish if contact is required for signaling and will ask if cells that make contact via filopodial extensions can signal. SPECIFIC AIM 4 The role of HSPGs in the distribution of cytonemes. We will determine whether cytonemes can extend over cells that lack HSPGs. These experiments will test whether inhibition of morphogen movement through regions lacking HSPGs correlates with the absence of cytonemes. SPECIFIC AIM 5 Cytoneme function. We will map cytonemes in imaginal discs that have ectopic signaling centers or that have reduced levels of Dpp at their A/P signaling center in order to examine the relationship between signaling and cytoneme orientation. We will also monitor Dpp signaling in discs whose cytonemes have been disrupted and we will track the movement of signaling proteins to determine if cytonemes function as conduits for signal traffic.